Mammalian-specific sequences in pou3f2 contribute to maternal behavior.

Various mutations have occurred during evolution among orthologs, genes in different species that diverged from a common ancestral gene by speciation. Here, we report the remarkable deterioration of a characteristic mammalian maternal behavior, pup retrieval, in nonmammalized mice, in which the transcription factor Pou3f2 was replaced with the Xenopus ortholog lacking all of the homopolymeric amino acid repeats of mammalian POU3F2. Most of the pups born to the nonmammalized mice died within days after birth, depending on the dam genotype alone. Quantitative immunohistochemical analysis revealed decreases in the rate-limiting enzymes of dopamine and serotonin synthesis in various brain structures. Similar results were obtained in knock-in mice in which all of the homopolymeric amino acid repeats of mammalian POU3F2 were removed. Pup retrieval behavior in mammals is thus strongly related to monoamine neurotransmitter levels via the acquisition of homopolymeric amino acid repeats during mammalian evolution.

Effect of different frequencies of music on blood pressure regulation in spontaneously hypertensive rats.

The effect of different frequencies of music on brain function was investigated through measurement of blood pressure in spontaneously hypertensive rats (SHR). Previous studies indicated that exposure to Mozart's music (K. 205) leads to increased calcium/calmodulin-dependent dopamine synthesis in the brain, and that the subsequent increase in dopamine reduces blood pressure via D(2) receptors. The present study demonstrated that the blood pressure-reducing response was dependent on the frequency, and was markedly greater at 4 k-16 kHz compared with lower frequencies. These findings suggest that music containing high-frequency sounds stimulates dopamine synthesis, and might thereby regulate and/or affect various brain functions.

Music improves dopaminergic neurotransmission: demonstration based on the effect of music on blood pressure regulation.

The mechanism by which music modifies brain function is not clear. Clinical findings indicate that music reduces blood pressure in various patients. We investigated the effect of music on blood pressure in spontaneously hypertensive rats (SHR). Previous studies indicated that calcium increases brain dopamine (DA) synthesis through a calmodulin (CaM)-dependent system. Increased DA levels reduce blood pressure in SHR. In this study, we examined the effects of music on this pathway. Systolic blood pressure in SHR was reduced by exposure to Mozart's music (K.205), and the effect vanished when this pathway was inhibited. Exposure to music also significantly increased serum calcium levels and neostriatal DA levels. These results suggest that music leads to increased calcium/CaM-dependent DA synthesis in the brain, thus causing a reduction in blood pressure. Music might regulate and/or affect various brain functions through dopaminergic neurotransmission, and might therefore be effective for rectification of symptoms in various diseases that involve DA dysfunction.

The significance of increase in striatal D(2) receptors in epileptic EL mice.

The present study systematically and quantitatively analyzed the immunohistochemical distribution of various substances involved in synthesis, binding, and transport of dopamine in the forebrain of epileptic mice (EL mouse strain) using a brain mapping analyzer. A reduction in serum calcium levels decreases calcium/calmodulin-dependent-dopamine synthesis in the brain and subsequently increases susceptibility to epileptic convulsions and induces abnormal behavior in EL mice. The immunohistochemical levels of D(2) receptors in the medial area of the neostriatum were significantly higher in EL mice than in ddY mice (mother strain of EL mice), while there were no differences in the levels of tyrosine hydroxylase, calcium/calmodulin-dependent protein kinase II, calmodulin, D(1) receptors, and dopamine transporters. Together with our previous findings, the results suggest that the decrease in serum calcium levels and subsequent decrease in brain dopamine synthesis comprise the primary physiologic disorder in EL mice, and convulsions or increased D(2) receptors are secondarily-induced phenomena to improve or compensate for the principal disorder.

Regulation of brain function by exercise.

The effect of excercise on brain function was investigated through animal experiments. Exercise leads to increased serum calcium levels, and the calcium is transported to the brain. This in turn enhances brain dopamine synthesis through a calmodulin-dependent system, and increased dopamine levels regulate various brain functions. There are abnormally low levels of dopamine in the neostriatum and nucleus accumbens of epileptic mice (El mice strain) and spontaneously hypertensive rats (SHR). The low dopamine levels in those animals were improved following intracerebroventricular administration of calcium chloride. Dopamine levels and blood pressure in SHR were also normalized by exercise. In epileptic El mice, convulsions normalized dopamine levels and physiologic function. These findings suggest that exercise or convulsions affect brain function through calcium/calmodulin-dependent dopamine synthesis. This leads to the possibility that some symptoms of Parkinson's disease or senile dementia might be improved by exercise.

Effect of nutritive and tonic crude drugs on physical fatigue-induced stress models in mice.

The present study was undertaken to investigate the acute anti-fatigue effect of a liquid nutritive and tonic crude drugs (NTDs) on stress induced in mice. After forced walking for 3 or 6h, the NTDs (applied orally, 10 ml/kg) significantly increased locomotor activity, while the administration of NTDs after rapid eye movement (REM) sleep deprivation stress and after immobilization stress did not show a specific effect, having a similar effect as the vehicle with added vitamins, taurine and caffeine. The administration of NTDs after freezing due to electric shock stress showed a specific effect which was not seen in other control groups, water, vehicle (ethanol) and vehicle including vitamins, taurine and caffeine and so resemble the specific effect of NTDs in the stress of forced walking. The present results indicate that the NTDs produced an anti-fatigue effect on the decreased locomotor activity after forced walking and immobility induced by electric stimulation. However, the crude drugs were not effective in improving immobility after sleep deprivation or immobilization stress.

A novel technique for quantitative immunohistochemical imaging of various neurochemicals in a multiple-stained brain slice.

Here we describe a novel technique for comparative analysis of the distributions of various neurochemicals visualized using multiple immunohistochemistry in the same brain slice. As an example, the distributions of tyrosine hydroxylase, substance P and glutamate decarboxylase in coronal slices of rat brains were compared. Each slice was divided into approximately 220,000-300,000 microareas at 20-microm intervals, and the immunohistochemical intensities of the three substances in each microarea were analyzed independently using a brain mapping analyzer; a microphotometry system previously developed in our laboratory (Sutoo et al., J. Neurosci. Methods, 1998; 85: 161-73). No significant differences between the distribution of each substance were observed in single- and triple-labeled slices. We believe that this method will facilitate the investigation of the functions of the central nervous system and the disorders thereof in various diseases.

Neurochemical changes in mice following physical or psychological stress exposures.

An investigation on the mechanism of neurochemical changes in physically or psychologically stressed mice was carried out. Physical stress was induced by electric foot shocks (2 mA for 5 s at 30-s intervals), and psychological stress was induced by emotional stimuli from electric foot-shocked mice using a communication box. The serum and brain calcium levels and immunohistochemical brain dopamine levels increased, and the ethanol-induced sleeping time was prolonged following exposure to these stimuli. The effects of electric foot shocks on these physiological parameters were greater than those of emotional stimuli. In the psychologically stressed mice, serum and brain calcium levels significantly increased 15 and 60 min, respectively, after the start of exposure to stimuli. Also, the immunohistochemical dopamine levels in the neostriatum and nucleus accumbens regions after 60 min of exposure to psychological stress were higher by 23% (P < 0.01) and 27% (P < 0.01), respectively, than those in unstressed control mice. Moreover, the ethanol-induced sleeping time was prolonged by approximately 60-100% (P < 0.01) in mice exposed to psychological stress for 30-120 min. The effect of emotional stimuli to prolong the ethanol-induced sleeping time was inhibited by intracerebroventricular administration of W-7 (a calmodulin antagonist) or alpha-methyltyrosine (an inhibitor of tyrosine hydroxylase). In light of previous reports that calcium activates dopamine synthesis in the brain via a calmodulin-dependent system, it is suggested that physical or psychological stimuli induce an increase in the brain calcium level, and this increased calcium level in turn enhances dopamine synthesis in the brain. Subsequently, an increased dopamine level induces various physiological changes related to stress-dependent phenomena.

Comparison analysis of distributions of tyrosine hydroxylase, calmodulin and calcium/calmodulin-dependent protein kinase II in a triple stained slice of rat brain.

The immunohistochemical distributions of tyrosine hydroxylase (TH), calmodulin (CaM) and calcium/CaM-dependent protein kinase II (CaMKII) in the rat forebrain were compared quantitatively to confirm our previous finding that TH activity and dopamine synthesis in the brain are regulated by a calcium/CaM-dependent system. The same slice was triply stained and the above substances were detected immunohistochemically. Their distributions in the slice were measured using a brain mapping analyzer which is a microphotometry system for the analysis of the distribution of neurochemicals in a large tissue slice. Each coronal section was divided into approximately 250000 to 310000 microareas at 20-microm intervals, and the immunohistochemical fluorescence intensities of the three substances in these microareas were analyzed independently. Quantitative images of the distributions were reconstructed from the data, and the distribution of each substance was investigated. Immunoreactive staining of TH, CaM and CaMKII was observed in almost all areas of the brain, but the intensities varied. Markedly intense TH-, CaM- and CaMKII-like immunoreactivities were distributed in the anterior dorsolateral and posterior areas of the neostriatum, nucleus accumbens and olfactory tubercle. In the previous study, the amount of dopamine was increased by the intracerebroventricular administration of calcium chloride in the neostriatum and nucleus accumbens. Combining these results with those previously reported, it is suggested that TH activity and dopamine synthesis in these regions are regulated by calcium ions via CaM and CaMKII. This method is a powerful technique for quantitative and comparative analysis of the distributions of various neurochemicals in the same slice, and we believe that it will facilitate investigation of the functions of the central nervous system and disorders thereof in various diseases.